Canto 2 flow cytometry system

SW1088 and SW1783 cell suspensions (3.0 × 104 cells/ml) were plated on 35 mm confocal dishes (Biosharp). Then, bromodeoxyuridine (BrdU) (Roche) with U73122, DMSO, or PBS was added to the medium. After 48 h, cells were fixed with 70% acidic ethanol for 20 min at 4 °C, followed by incubation with primary anti-BrdU antibody (ab6326, Abcam), then Alexa-488 secondary antibody, and finally DAPI. BrdU- and DAPI-positive cells were then counted in ImageJ.
For cell cycle analysis, cells were serum starved for 24 h, and then the medium was replaced with complete growth medium. After 12 or 24 h, cells were collected for DNA staining using propidium iodide (PI), and DNA content was analysed by using a BD Canto II Flow Cytometry System (BD Biosciences) and FlowJo software, v10.6.
For the apoptosis assay, cells were serum starved as described above and then freshly collected and stained for early apoptosis markers using a fluorescein isothiocyanate (FITC) annexin V apoptosis detection kit (BD Biosciences) according to the manufacturer’s instructions. Annexin V and PI staining were measured using a flow cytometer and analysed using FlowJo v10.6.

First, the peripheral blood was diluted and mixed with RPIM1640 (Gibco, Germany) at a ratio of 1:1, and then gently layered on top of 4 mL of Ficoll-Hypaque 10,771 (Sigma, USA). The tubes were centrifugated at 1600 rpm for 30 min at 25 °C. Then, the PBMC layer was aspirated and transferred into a new fresh 15 mL conical centrifuge tube. The PBMCs were mixed with RPIM1640 to wash twice at 1300 rpm for 10 min. After cell counting, fresh PBMCs were stained with different FCM antibodies and tested using a BD Canto II Flow Cytometry System. The remaining PBMCs were resuspended and cryopreserved with freezing media, including 10% dimethysulfoxide (DMSO) and 90% fetal bovine serum. The cell number was generally at least 5 × 10⁶ per tube. Lastly, the frozen PBMC samples were transported into a liquid nitrogen container for long-time preservation after being stored in the freezing container at − 80 °C overnight. 5–10 samples were processed at a time and the subsequent freez thaw process followed every step of the protocol strictly.